The pathophysiology of nonalcoholic fatty liver disease (NAFLD) was characterized by alterations in the intestinal microbiota and bile acids (BAs). Flaxseed powder (FLA) was rich in active components such as α-linolenic acid, dietary fiber, and lignans, which had lipid-lowering and anti-inflammatory effects. Here, we investigated whether FLA reduced liver fat and improved inflammation by modulating the gut microbiota, enriching bacteria involved in the production of 6α-hydroxylated BAs, and activating the gut-liver-BA metabolic pathway-specific receptor Takeda G protein-coupled receptor 5 (TGR5). Wild-type (WT) and TGR5 knockout mice were set up in a low-fat control group, a high-fat model group and a flaxseed powder intervention group for 12 weeks. At the end of the experiment, we examined the levels of lipids (TC, LDL-C, HDL-C, and TG), the levels of inflammatory factors (TNF-α and IL-6), the pathological changes in the liver, and the differences in the expression of key proteins (CYP7A1, TLR4, and NF-κB) in the liver of TGR5–/– and WT mice. In the current study, we found that 12 weeks of FLA intervention significantly attenuated the progression of NAFLD in WT mice, whereas TGR5 knockout exacerbated the extent of disease in mice with NAFLD. TGR5 gene knockout blocked the anti-inflammatory effect of FLA, but did not block its lipid-lowering effect. The TGR5 gene may be a key protein in the anti-inflammatory pathway of FLA, rather than a key protein in the lipid-lowering pathway of FLA. FLA intervention altered the relative abundance of gut microbiota with BA metabolizing enzymes, which in turn regulated the composition of intestinal BA, particularly the proportion of the key functional BAs 6α-hydroxylated BAs, thereby activating the intestinal BA-specific receptor TGR5, which might play a role in ameliorating inflammation. FLA might be a promising functional food for the prevention of NAFLD by modulating the microbiota and BAs.